High throughput platform technology for rapid target identification in personalized phage therapy

As bacteriophages continue to gain regulatory approval for personalized human therapy against antibiotic-resistant infections, there is a need for transformative technologies for rapid target identification through multiple, large, decentralized therapeutic phages biobanks. Here, we design a high throughput phage screening platform comprised of a portable library of individual shelf-stable, ready-to-use phages, in all-inclusive solid tablets. Each tablet encapsulates one phage along with luciferin and luciferase enzyme stabilized in a sugar matrix comprised of pullulan and trehalose capable of directly detecting phage-mediated adenosine triphosphate (ATP) release through ATP bioluminescence reaction upon bacterial cell burst. The tablet composition also enhances desiccation tolerance of all components, which should allow easier and cheaper international transportation of phages and as a result, increased accessibility to therapeutic phages. We demonstrate high throughput screening by identifying target phages for select multidrug-resistant clinical isolates of Pseudomonas aeruginosa, Salmonella enterica, Escherichia coli, and Staphylococcus aureus with targets identified within 30-120 min.

Supplementary Figure 1.Optical density (OD600) or growth curve of Pseudomonas aeruginosa PAO1 strain (Pa).a Pa infected with P32 shows a strong performance in preventing bacterial growth by decreasing the culture turbidity even at lower MOIs (0.001).b Pa infected with JG004 showed effective bacterial growth suppression c Pa infected with PP7 showed week efficacy is preventing bacterial growth which aggravates by decreasing the initial concentration of phage (lower MOIs).The represented data are the average of three independent experiments (n=3) with at least 6 technical replicates.Source data are provided in Source Data file.Supplementary Figure 2. Schematics of phage life cycles.a Lytic life cycle.The lytic cycle of phages starts with (i) phage recognition and attachment to bacterial cell receptors, (ii) genome injection, (iii) taking control of the bacterial replication machinery and producing new virions, and (iv) release of progeny phages by bacterial cell lysis.b Lysogenic life cycle.In the lysogenic life cycle, phage also adsorbs to cell receptors (i) and injects its genome inside the bacterial cell (ii); however, the phage genome is integrated into the bacterial genome, forming a prophage (iii) that is replicated through the bacterial reproduction cycle (iv).The lysogenic cycle could be induced to start the lytic cycle with environmental triggers such as high temperature or UV exposure which can ultimately lead to cell lysis (as shown by the dashed arrow).c Chronic life cycle.Similar to other phage life cycles, the chronic lifestyle (mostly seen in filamentous phages) starts with recognition and adsorption to bacterial cell receptors (i) and genome injection (ii).By taking control of the bacterial replication machinery, new virions are synthesized inside the bacterial cell (iii) and are released through budding or extrusion without lysing bacterial cells (iv).Some chronic phages can also adopt a lysogenic lifestyle, in which the phage genome is incorporated into the bacterial genome forming a prophage (iii′) and is replicated through the bacterial reproduction Supplementary Figure 4. Effect of sugar polymers on stability of the ATP reagent solution at 37°C.ATP standard solutions were added to ATP reagent solution with (+) and without (-) sugar mixture at time =0, after incubating for one, three and six hours at 37°C, and the results were compared to no heat exposure ATP reagent solution.ATP standard solutions are used at a 0.4 µM, b 0.01 µM, and c 0.001 µM.The RLU signal was measured kinetically every 10 minutes for up to 3 hours.Sugar mixture was able to preserve the activity of luciferin and luciferase as there were no significant changes in the RLU signals after heat exposures.In the absence of sugars, the activity of ATP reagent solution decreased as significantly lower RLU signals were detected, which was more drastic after 3 and 6 hours of heat exposure.The RLU signal also dropped faster in the absence of sugar mixture.The presented data are the average of three replicates (n=3) with error bars representing standard deviation from the mean.Source data are provided in Source Data file.Supplementary Figure 8. Screening Clinical P. aeruginosa isolates against an in-house phage library.a C0072 strain isolated from a patient with urinary tract infection screened against a library of phages including phages from different species (Supplementary Table 1) using desiccated Sugar-based one-pot ATP bioluminescence assay, optical density assay (OD600), and representative spot test on C0072 bacterial lawn.b C0335 strain isolated from a patient with arm infection, screened against an in-house phage library using one-pot ATP bioluminescence assay optical density assay (OD600) and spot test on C0335 bacterial lawn.The OD600 assay and spot test was conducted on three P. aeruginosa phages including P32, JG004, and PP7.ATP and OD assay were conducted in triplicates (n=3).The dashed lines show standard deviation from the mean in OD600 assays and the error bars in ATP assays are standard deviation from the mean.Source data are provided in Source Data file.10.Zoomed in image of the phage library screening, showing a delayed increase in bioluminescence signal for C0035 infected with JG004 appearing after 2 hours.The ATP assay was conducted in triplicates (n=3).The dashed lines show standard deviation from the mean.Source data are provided in Source Data file.Supplementary Figure 12.ATP and OD600 kinetic curves and spot test of two E. coli strains against 28 E. coli phages.a E. coli O157:H7 strain (human fecal isolate).b LF82 (Crohn's disease isolate 1 ).ATP one-pot assay was conducted in the presence of sugar polymers.The ATP and OD600 assays were conducted in triplicates (n=3).The error bars show standard deviation from the mean.Source data are provided in Source Data file.10 −8 (~5 × 10 11 PFU/mL to ~5 × 10 3 PFU/mL).For the ATP and OD600 assays, bacterial subcultures were infected with phage JG004 at MOIs of 10,000, 1000, 100, 10, 1, 0.1, 0.01.Panel a) Pa:JG004, b) C0072:JG004, c) C0335:JG004.The ATP and OD600 assays were conducted in triplicates (n=3).The error bars show standard deviation from the mean.Source data are provided in Source Data file.+++: strong signal (ATP assay: the highest and continuous RLU signal detected upon strain infection within the phage library, OD600: the lowest turbidity detected upon strain infection within the phage library in comparison with negative control (uninfected strain), Spot test: clear plaque on the strain's bacterial lawn) ++: medium signal (ATP assay: a medium RLU signal detected upon strain infection in comparison with the highest RLU value within the phage library, OD600: the medium reduction in turbidity detected upon strain infection in comparison with negative control, Spot test: turbid plaque on the strain's bacterial lawn) +: weak signal (ATP assay: a weak RLU signal value detected upon strain infection in comparison with the highest and medium RLU value within the phage library, OD600: a weak reduction in turbidity detected upon strain infection in comparison with negative control, Spot test: faint plaque on the strain bacterial lawn : very faint footprint Supplementary Table 6 +++: strong signal (ATP assay: the highest and continuous RLU signal detected upon strain infection within the phage library, OD600: the lowest turbidity detected upon strain infection within the phage library in comparison with negative control (uninfected strain), Spot test: clear plaque on the strain's bacterial lawn) ++: medium signal (ATP assay: a medium RLU signal detected upon strain infection in comparison with the highest RLU value within the phage library, OD600: the medium reduction in turbidity detected upon strain infection in comparison with negative control, Spot test: turbid plaque on the strain's bacterial lawn) +: weak signal (ATP assay: a weak RLU signal value detected upon strain infection in comparison with the highest and medium RLU value within the phage library, OD600: a weak reduction in turbidity detected upon strain infection in comparison with negative control, Spot test: faint plaque on the strain bacterial lawn : very faint footprint

Supplementary
cycle and staying dormant (iv′) until the chronic cycle is induced by environmental stimuli (shown by the dashed arrow).Panels a and b Created with BioRender.comreleased under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.Supplementary Figure 3. Decreasing ATP background in phage solutions.a ATP background at different stages of P32 phage purification.PEG purification decreased the ATP background in phage solution.10 KDa ultrafiltration decreased the ATP background significantly, while 3 KDa filters failed to reduce ATP levels.b Effect of dilution of phage suspension in bacterial cell culture media (LB media) on ATP background.The effect of diluting phage samples is comparable to purifying phage suspensions.Depending on the availability, either diluted filtered phages in bacterial cell media/buffer or purified phages can be used to reduce the effect of the background bioluminescence signal intensity.Results shown are the average of three replicates (n=3) with associated error bars showing standard deviation from the mean.Source data are provided in Source Data file.

Figure 5 .
Comparison of the RLU signal intensity of heat treated and untreated ATP reaction solutions in the presence and absence of sugar mixture after heat exposure at 37°C.RLU signal at time=0, right after adding ATP standard solution with 0.4 µM, 0.01 µM, 0.001 µM to ATP reagent solutions with (+) and without (-) sugar mixture after incubating for 1, 3 and 6 hours at 37°C in comparison with no heat exposure ATP reagent solution (time=0).The presented data are the average of three replicates (n=3) with Standard deviation from the mean.Statistical significance in all panels is derived from Two-way analysis of variance (ANOVA).Significance levels include *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.Source data are provided in Source Data file.Supplementary Figure 6.Stability of dried Pseudomonas phages P32 (a), JG004 (b), and PP7 (c) in ambient conditions in 10 wt% pullulan + 0.5 M trehalose in comparison with no sugars.Sugar polymers helped to retain higher infectivity after 30 days.The presented data are the average of three replicates (n=3) with Standard deviation from the mean.Source data are provided in Source Data file.

Table 1 .
List of bacterial species and strains Clinical isolates from the in-house library at the Michael DeGroote Institute of Infectious Disease Research.* * From the Felix D'Herelle Reference Center for Bacterial Viruses Supplementary

Table 2 .
List of S. enterica phages

Table 5 .
Comparative rating of the ATP detection method with culture techniques for Salmonella phages

.
Comparative rating of the ATP detection method with culture techniques for E. coli phages ATP assay: a medium RLU signal detected upon strain infection in comparison with the highest RLU value within the phage library, OD600: the medium reduction in turbidity detected upon strain infection in comparison with negative control, Spot test: turbid plaque on the strain's bacterial lawn) +: weak signal (ATP assay: a weak RLU signal value detected upon strain infection in comparison with the highest and medium RLU value within the phage library, OD600: a weak reduction in turbidity detected upon strain infection in comparison with negative control, Spot test: faint plaque on the strain bacterial lawn : The kill curve and ATP detection methods exhibit relatively strong signals, leading to the conclusion that the spot test in this case may be biased.

Table 7 .
Comparative rating of the ATP detection method with culture techniques for S. aureus phages